Field of the Invention
The invention relates to a method for carrying out recombination at a target locus in a Rasamsonia cell. The invention also relates to Rasamsonia cells, for example Rasamsonia cells produced by such a process. The invention further relates to processes in which such Rasamsonia cells are used and to the resulting enzyme compositions. The invention further relates to nucleic acid and amino acid sequences.
Description of Related Art
Carbohydrates constitute the most abundant organic compounds on earth. However, much of this carbohydrate is sequestered in complex polymers including starch (the principle storage carbohydrate in seeds and grain), and a collection of carbohydrates and lignin known as lignocellulose. The main carbohydrate components of lignocellulose are cellulose, hemicellulose, and pectins. These complex polymers are often referred to collectively as lignocellulose.
Bioconversion of renewable lignocellulosic biomass to a fermentable sugar that is subsequently fermented to produce alcohol (e.g., ethanol) as an alternative to liquid fuels has attracted an intensive attention of researchers since 1970s, when the oil crisis broke out because of decreasing the output of petroleum by OPEC. Ethanol has been widely used as a 10% blend to gasoline in the USA or as a neat fuel for vehicles in Brazil in the last two decades. More recently, the use of E85, an 85% ethanol blend has been implemented especially for clean city applications. The importance of fuel bioethanol will increase in parallel with increases in prices for oil and the gradual depletion of its sources. Additionally, fermentable sugars are being used to produce plastics, polymers and other biobased products and this industry is expected to grow substantially therefore increasing the demand for abundant low cost fermentable sugars which can be used as a feed stock in lieu of petroleum based feedstocks.
The sequestration of such large amounts of carbohydrates in plant biomass provides a plentiful source of potential energy in the form of sugars, both five carbon and six carbon sugars that could be utilized for numerous industrial and agricultural processes. However, the enormous energy potential of these carbohydrates is currently under-utilized because the sugars are locked in complex polymers, and hence are not readily accessible for fermentation. Methods that generate sugars from plant biomass would provide plentiful, economically-competitive feedstocks for fermentation into chemicals, plastics, such as for instance succinic acid and (bio) fuels, including ethanol, methanol, butanol synthetic liquid fuels and biogas.
Regardless of the type of cellulosic feedstock, the cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of the biomass bioconversion processes. The production costs of microbially produced enzymes are tightly connected with a productivity of the enzyme-producing strain and the final activity yield in the fermentation broth.
In spite of the continued research of the last few decades to understand enzymatic lignocellulosic biomass degradation and cellulase production, it remains desirable to discover or to engineer new highly active cellulases and hemicellulases. It would also be highly desirable to construct highly efficient enzyme compositions capable of performing rapid and efficient biodegradation of lignocellulosic materials.
Such enzyme compositions may be used to produce sugars for fermentation into chemicals, plastics, such as for instance succinic acid and (bio) fuels, including ethanol, methanol, butanol, synthetic liquid fuels and biogas, for ensiling, and also as enzyme in other industrial processes, for example in the food or feed, textile, pulp or paper or detergent industries and other industries.
One genus of microorganisms that is known to produce suitable enzymes for enzymatic lignocellulosic biomass degradation is the genus Rasamsonia. Rasamsonia is a filamentous fungus and is sometimes referred to as Talaromyces. 
Jain, S. et al, Mol Gen Genet (1992), 234, 489-493 discloses a transformation system for the fungus Talaromyces sp CL240. No expression of polypeptides is disclosed.
Murray, F. R. et al, Curr Genet (1997), 32, 367-375 discloses over-expression of the glucose oxidase gene from Talaromyces flavus in Talaromyces macrosporus. The effect fungal isolates on growth inhibition of V. dahliae was studied.
WO200170998 discloses Talaromyces emersonii beta-glucanases. On page 16, it is described that the polynucleotide of beta-glucanase may be heterologously expressed in a host, e.g. a yeast cell.
WO200224926 discloses Talaromyces emersonii xylanase. On page 24, 5th paragraph, it is described that production of the polypeptide may be achieved by recombinant expression of the xylanase DNA sequence in a suitable homologous or heterologous host cell. In paragraph 7, it is said that the host cell may over-express the polypeptide, and techniques for engineering over-expression are well known from WO99/32617. WO99/32617 relates to expression cloning, but does not disclose cloning in Talaromyces host.
WO2007091231 discloses strains of Talaromyces emersonii which are thermostable and encode thermostable enzymes, and also discloses enzyme compositions produced by the Talaromyces emersonii strains. No recombinant production of homologous or heterologous polypeptides is disclosed. In table 1 shows inducing carbon sources were added in an amount of 0.2 to 6%. Solka floc and glucose (2%) were included for comparative purposes. On page 78, line 28 it is said that “glucose does not completely repress exoglucosidase production by the T. emersonii strains (table 31A). Table 31A shows that IM1393751 produces beta-glucosidase activity of 31.90 IU with glucose as carbon source, but no other cellulase activities, e.g. glucanases or xylanases. Due to lack of such enzyme activities, the strain IM1393751 is not suitable for the production of cellulases for the conversion of lignocellulose on glucose as carbon source.
WO2011054899 discloses Talaromyces transformants and a process for production of polypeptides using the Talaromyces transformants. Transformants in which polynucleotides of interest are introduced, are selected using a selection marker such as the phleomycin resistance marker, which is introduced at the same time as the polynucleotide of interest. Such stable hosts will therefore contain a selection marker.
Additional genetic tools are required so that Rasamsonia may be used more effectively for the production of enzymes or other industrially relevant products.